ABSTRACT This exploratory R21 proposal describes two novel nanotechnologies for acidification of the lysosome and for restoration of autophagic flux. Autophagy, the intracellular process by which proteins and organelles are degraded, requires an acidic lysosome. Failure to acidify the lysosomal compartment affords accumulation of autophagosomes, lipids, and associated undigested content. Impaired lysosomal acidification and reduced autophagic flux are central to non-alcoholic fatty liver disease (NAFLD). NAFLD occurs primarily among the middle-aged and the elderly given that the risk factors for its development increase with advancing age. Current pharmacological and molecular tools are able to reduce lysosomal acidity (increase pH); however, no tools exist to increase lysosomal acidity (decrease pH). Key to our advance is the use of lysosomal accumulating nanoparticles (NPs) that release acid to lower the local pH. Specifically, we propose two new NP technologies to increase lysosomal acidity. The first is a stimuli-responsive NP that acidifies its local environment upon exposure to light (i.e., light activated acid nanoparticle, light-acNP). The second NP responds to the pH of a dysfunctional lysosome (5.7 - 6) to release acid further lowering the pH to a healthy/normal value (4 - 5). These pH-activated acid nanoparticles (pH-acNPs) are polyester-based NPs composed of a potent diacid, tetrafluorosuccinic acid (TFSA), to further lower the pH upon NP degradation. We hypothesize that upon accumulation in lysosomes, the acNPs will release acid through light activation or pH-activation and thereby restore lysosomal acidity and autophagic flux quickly. Further, we hypothesize that the extent of restoration will depend on the length of light exposure and the amount of TFSA in the polymer, respectively. Importantly, substantial preliminary data support the proposed studies, well-characterized materials and rigorous experimental designs are established, and essential cross-disciplinary collaborations and expertise (nanotechnology, polymer chemistry, cell metabolism, and autophagy) are in place to address these hypotheses. The specific aims of this two-year proposal are: Aim 1. Evaluate lysosome-targeted acNPs that activate with light (light-acNPs); and, Aim 2. Synthesize and evaluate lysosome-targeted acNPs that activate at pH 6 (pH-acNPs). Successful completion of the proposal will provide two new tools for scientists, clinicians, and biomedical engineers to study autophagy biology to facilitate agent screening and mechanism(s) of action. From a nanotechnology perspective, new nanoparticle compositions and functions will be identified as well as a means to control a key cellular process via a nanoparticle. Finally, these results would support further evaluation of the acNPs in in vivo murine models to include safety, PK/PD, and efficacy studies as part of a future R01 proposal focused on NAFLD.